1. Field of the Invention
This invention relates to a method and composition for protecting mammalian cells, being preserved by lyophilization. More particularly, the invention pertains to a method and composition for introducing protective solutes into red blood cells to preserve the cell during lyophilization and for increasing the retention of useful cells on rehydration.
2. Description of the Prior Art
The preservation of labile proteins in the dry state by carbohydrates has been well documented (Bonderman, D. P., Proksch, G. J., and Haskins, S., "A Lyophilized Hemoglobin Control Prepared From Stroma-free Hemolysates," Clin. Chem., 26:2, 305-308, (1980); DeVenuto, F., Zegna, A. I., and Busse, K. R., "Lyophilization of Crystalline Hemoglobin Solution and Exchange Transfusions with Lyophilized, Reconstituted Hemoglobin," Surgery, Gynecology, and Obstetrics, 148, 69-75, (1979)). The recovery of functional hemoglobin (Hb) following lyophilization has been observed with a variety of carbohydrates (Thirion, C., Larcher, D., Chaillot, B., Labrude, P., and Vigneron, C., "Circular Dichroism Studies of Freeze-dried Induced Conformational Changes in Human Hemoglobin," Biopolymers, 22, 2367-2381, (1983)). The disaccharide trehalose has been shown in this work to be equally effective in retaining the functional oxygen binding characteristics of Hb as well as inhibiting oxidation of Hb to methemoglobin (metHb) in the dry state. The most likely mechanism of damage to proteins in the dry state is by free radical damage. Deleterious radicals accumulate in organisms that are damaged by severe dehydration, while those that accumulate protective carbohydrates maintain low levels of free radicals (Heckly, R. J., Effects of Oxygen on Dried Organisms in "Dry Biological Systems", (J. Crowe, ed.), 257-279, Academic Press, New York, 1978). One of the ways carbohydrates may protect biological systems from oxidative damage in the dry state is to act as scavengers of radicals that can oxidize essential biomacromolecules, Heckly supra. In these experiments, cold storage of lyophilized Hb (after rehydration) showed the rate of oxidative damage to Hb (as measured by the increase in metHb) is approximately the same as in those samples with added carbohydrates. This suggests that the protective antioxidant action of carbohydrates may be restricted to the dry state. This may be due to the half-life of the radical as it is thought to be much more stable in the absence of bulk water, Heckly supra. Another consideration is the use of these carbohydrates as substrates for bacterial growth in hydrated samples stored at 4.degree. C. The presence of increasing amounts of bacterial endotoxins will also result in accelerated oxidation of Hb (unpublished data).
The ability of carbohydrates to maintain cell size during lyophilization is correlated to the ability of carbohydrates to inhibit dehydration. Previous work has demonstrated that trehalose, sucrose, and glucose (to a lesser degree) inhibit dehydration. This action may be due to the binding of carbohydrates to a cell wall or to a liposome (Crowe, L. M., Crowe, J. H., Rudolph, A. S., Womersley, C., and Appel, L., "Preservation of Freeze-dried Liposomes by Trehalose," Arch. Biochem. Biophys., 242:1, 240-247, (I985); Crowe, J. H., and Crowe, L. M., "Effects of Dehydration on Membranes and Membrane Stabilization at Low Water Activities," In Biological Membranes, (D. Chapman, Ed.), Vol. 5, Academic Press, New York/London, 1985; Rudolph, A. S., and Crowe, J. H., "A Calorimetric and Infrared Spectroscopic Study of the Stabilizing Solute Proline," Biophys. J., 50, 423-430 (1986)).
Baldeschwieler et al., in U.S. Pat. No.4,915,951, provides a summary of articles and patents relating to the discovery of and development of trehalose as a cryopreservation agent. Baldschwieler et al. discloses a lipophilic anchor molecule to assist in introducing a carbohydrate to the membrane wall. Disaccharides, such as maltose, are among the polyoxygen containing compounds which Baldeschwieler suggests as part of the cryopreservation agent.
Horan et al., in U.S. Pat. No. 4,783,401, discloses the use of osmolarity regulating agents to assist in introducing marking dyes into cells including blood cells. The suggested osmolarity regulating agents include sugars, sugar-alcohols, amino acids and certain hydrogen ion buffers known as "Good's Buffers". These osmolarity regulating agents include monosaccharides and disaccharides including glycerol, inositol and the like.
While freeze-thawing is now an accepted method of preserving a variety of mammalian cell lines, currently, there are no satisfactory methods of preserving red blood cells in the dry state. These red blood cells are inherently unstable to reduced water conditions because of oxidation events (expressed as generation of methemoglobin, the oxidative degeneration of hemoglobin) and destabilization of the cell membrane.